Comprehensive Identification of the Pum Gene Family and Its Involvement in Kernel Development in Maize.

The Pumilio (Pum) RNA-binding protein family regulates post-transcription and plays crucial roles in stress response and growth. However, little is known about Pum in plants. In this study, a total of 19 ZmPum genes were identified and classified into two groups in maize. Although each ZmPum contains the conserved Pum domain, the ZmPum members show diversity in the gene and protein architectures, physicochemical properties, chromosomal location, collinearity, cis-elements, and expression patterns. The typical ZmPum proteins have eight α-helices repeats, except for ZmPum2, 3, 5, 7, and 14, which have fewer α-helices. Moreover, we examined the expression profiles of ZmPum genes and found their involvement in kernel development. Except for ZmPum2, ZmPum genes are expressed in maize embryos, endosperms, or whole seeds. Notably, ZmPum4, 7, and 13 exhibited dramatically high expression levels during seed development. The study not only contributes valuable information for further validating the functions of ZmPum genes but also provides insights for improvement and enhancing maize yield.

Maize/soybean strip intercropping produces higher crop yields and saves water under semi-arid conditions.

Sustainable increases in crop production require efficient use of resources, and intercropping can improve water use efficiency and land productivity at reduced inputs. Thus, in a three-year field experiment, the performance of maize/soybean strip intercropping system differing with maize plant density (6 maize plants m-2, low, D1; 8 maize plants m-2, medium, D2; and 10 maize plants m-2, high, D3) was evaluated in comparison with sole maize or soybean cropping system. Results revealed that among all intercropping treatments, D2 had a significantly higher total leaf area index (maize LAI + soybean LAI; 8.2), total dry matter production (maize dry matter + soybean dry matter; 361.5 g plant-1), and total grain yield (maize grain yield + soybean grain yield; 10122.5 kg ha-1) than D1 and D3, and also higher than sole maize (4.8, 338.7 g plant-1, and 9553.7 kg ha-1) and sole soybean (4.6, 64.8 g plant-1, and 1559.5 kg ha-1). The intercropped maize was more efficient in utilizing the radiation and water, with a radiation use efficiency of 3.5, 5.2, and 4.3 g MJ-1 and water use efficiency of 14.3, 16.2, and 13.3 kg ha-1 mm-1, while that of intercropped soybean was 2.5, 2.1, and 1.8 g MJ-1 and 2.1, 1.9, and 1.5 kg ha-1 mm-1 in D1, D2, and D3, respectively. In intercropping, the land and water equivalent ratios ranged from 1.22 to 1.55, demonstrating that it is a sustainable strategy to improve land and water use efficiencies; this maximization is likely associated with the species complementarities for radiation, water, and land in time and space, which resulted in part from competition avoidance responses that maximize the economic profit (e. g., 1300 US $ ha-1 in D2) over sole maize (798 US $ ha-1) or sole soybean (703 US $ ha-1). Overall, these results indicate that optimizing strip intercropping systems can save 20-50% of water and land, especially under the present scenario of limited resources and climate change. However, further research is required to fully understand the resource capture mechanisms of intercrops in intercropping.

Genome-Wide Identification and In Silico Analysis of ZF-HD Transcription Factor Genes in Zea mays L.

Zinc finger-homeodomain proteins are amongst the most prominent transcription factors (TFs) involved in biological processes, such as growth, development, and morphogenesis, and assist plants in alleviating the adverse effects of abiotic and biotic stresses. In the present study, genome-wide identification and expression analyses of the maize ZHD gene family were conducted. A total of 21 ZHD genes with different physicochemical properties were found distributed on nine chromosomes in maize. Through sequence alignment and phylogenetic analysis, we divided ZHD proteins into eight groups that have variations in gene structure, motif distribution, and a conserved ZF domain. Synteny analysis indicated duplication in four pairs of genes and the presence of orthologues of maize in monocots. Ka/Ks ratios suggested that strong pure selection occurred during evolution. Expression profiling revealed that the genes are evenly expressed in different tissues. Most of the genes were found to make a contribution to abiotic stress response, plant growth, and development. Overall, the evolutionary research on exons and introns, motif distributions, and cis-acting regions suggests that these genes play distinct roles in biological processes which may provide a basis for further study of these genes' functions in other crops.

Nitrogen fertilization coupled with iron foliar application improves the photosynthetic characteristics, photosynthetic nitrogen use efficiency, and the related enzymes of maize crops under different planting patterns.

Photosynthetic rate (Pn) and photosynthetic nitrogen use efficiency (PNUE) are the two important factors affecting the photosynthesis and nutrient utilization of plant leaves. However, the effect of N fertilization combined with foliar application of Fe on the Pn and PNUE of the maize crops under different planting patterns (i.e., monocropping and intercropping) is elusive. Therefore, this experiment was conducted to determine the effect of N fertilization combined with foliar application of Fe on the photosynthetic characteristics, PNUE, and the associated enzymes of the maize crops under different planting patterns. The results of this study showed that under intercropping, maize treated with N fertilizer combined with foliar application of Fe had not only significantly (p < 0.05) improved physio-agronomic indices but also higher chlorophyll content, better photosynthetic characteristics, and related leaf traits. In addition, the same crops under such treatments had increased photosynthetic enzyme activity (i.e., rubisco activity) and nitrogen metabolism enzymes activities, such as nitrate reductase (NR activity), nitrite reductase (NiR activity), and glutamate synthase (GOGAT activity). Consequently, intercropping enhanced the PNUE and soluble sugar content of the maize crops, thus increasing its yield compared with monocropping. Thus, these findings suggest that intercropping under optimal N fertilizer application combined with Fe foliation can improve the chlorophyll content and photosynthetic characteristics of maize crops by regulating the associated enzymatic activities. Consequently, this results in enhanced PNUE, which eventually leads to better growth and higher yield in the intercropping system. Thus, practicing intercropping under optimal nutrient management (i.e., N and Fe) could be crucial for better growth and yield, and efficient nitrogen use efficiency of maize crops.

Genome-wide analyses of the mung bean NAC gene family reveals orthologs, co-expression networking and expression profiling under abiotic and biotic stresses.

BACKGROUND: Mung bean is a short-duration and essential food crop owing to its cash prominence in Asia. Mung bean seeds are rich in protein, fiber, antioxidants, and phytonutrients. The NAC transcription factors (TFs) family is a large plant-specific family, participating in tissue development regulation and abiotic and biotic stresses. RESULTS: In this study, we perform genome-wide comparisons of VrNAC with their homologs from Arabidopsis. We identified 81 NAC transcription factors (TFs) in mung bean genome and named as per their chromosome location. A phylogenetic analysis revealed that VrNACs are broadly distributed in nine groups. Moreover, we identified 20 conserved motifs across the VrNACs highlighting their roles in different biological process. Based on the gene structure of the putative VrNAC and segmental duplication events might be playing a vital role in the expansion of mung bean genome. A comparative phylogenetic analysis of mung bean NAC together with homologs from Arabidopsis allowed us to classify NAC genes into 13 groups, each containing several orthologs and paralogs. Gene ontology (GO) analysis categorized the VrNACs into biological process, cellular components and molecular functions, explaining the functions in different plant physiology processes. A gene co-expression network analysis identified 173 genes involved in the transcriptional network of putative VrNAC genes. We also investigated how miRNAs potentially target VrNACs and shape their interactions with proteins. VrNAC1.4 (Vradi01g03390.1) was targeted by the Vra-miR165 family, including 9 miRNAs. Vra-miR165 contributes to leaf development and drought tolerance. We also performed qRT-PCR on 22 randomly selected VrNAC genes to assess their expression patterns in the NM-98 genotype, widely known for being tolerant to drought and bacterial leaf spot disease. CONCLUSIONS: This genome-wide investigation of VrNACs provides a unique resource for further detailed investigations aimed at predicting orthologs functions and what role the play under abiotic and biotic stress, with the ultimate aim to improve mung bean production under diverse environmental conditions.

Identification, Association of Natural Variation and Expression Analysis of ZmNAC9 Gene Response to Low Phosphorus in Maize Seedling Stage.

Phosphorus (P) is an essential macroelement supporting maize productivity and low-P stress is a limiting factor of maize growth and yield. Improving maize plant tolerance to low P through molecular breeding is an effective alternative to increase crop productivity. In this study, a total of 111 diverse maize inbred lines were used to identify the favorable alleles and nucleotide diversity of candidate ZmNAC9, which plays an important role in response to low P and regulation in root architecture. A significant difference was found under low- and sufficient-P conditions for each of the 22 seedling traits, and a total of 41 polymorphic sites including 32 single nucleotide polymorphisms (SNPs) and 9 insertion and deletions (InDels) were detected in ZmNAC9 among 111 inbred lines. Among the 41 polymorphic studied sites, a total of 39 polymorphic sites were associated with 20 traits except for the dry weight of shoots and forks, of which six sites were highly significantly associated with a diverse number of low-P tolerant root trait index values by using a mixed linear model (MLM) at -log10 P = 3.61. In addition, 29 polymorphic sites under P-sufficient and 32 polymorphic sites under P-deficient conditions were significantly associated with a diverse number of seedling traits, of which five polymorphic sites (position S327, S513, S514, S520, and S827) were strongly significantly associated with multiple seedling traits under low-P and normal-P conditions. Among highly significant sites, most of the sites were associated with root traits under low-P, normal-P, and low-P trait index values. Linkage disequilibrium (LD) was strong at (r2 > 1.0) in 111 inbred lines. Furthermore, the effect of five significant sites was verified for haplotypes in 111 lines and the favorable allele S520 showed a positive effect on the dry weight of roots under the low-P condition. Furthermore, the expression pattern confirmed that ZmNAC9 was highly induced by low P in the roots of the P-tolerant 178 inbred line. Moreover, the subcellular localization of ZmNAC9 encoded by protein was located in the cytoplasm and nucleus. Haplotypes carrying more favorable alleles exhibited superior effects on phenotypic variation and could be helpful in developing molecular markers in maize molecular breeding programs. Taken together, the finding of this study might lead to further functions of ZmNAC9 and genes that might be involved in responses to low-P stress in maize.

Maize ZmBES1/BZR1-5 Decreases ABA Sensitivity and Confers Tolerance to Osmotic Stress in Transgenic Arabidopsis.

The BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1 (BZR1) transcription factors, key components in the brassinosteroid signaling pathway, play pivotal roles in plant growth and development. However, the function of BES1/BZR1 in crops during stress response remains poorly understood. In the present study, we characterized ZmBES1/BZR1-5 from maize, which was localized to the nucleus and was responsive to abscisic acid (ABA), salt and drought stresses. Heterologous expression of ZmBES1/BZR1-5 in transgenic Arabidopsis resulted in decreased ABA sensitivity, facilitated shoot growth and root development, and enhanced salt and drought tolerance with lower malondialdehyde (MDA) content and relative electrolyte leakage (REL) under osmotic stress. The RNA sequencing (RNA-seq) analysis revealed that 84 common differentially expressed genes (DEGs) were regulated by ZmBES1/BZR1-5 in transgenic Arabidopsis. Subsequently, gene ontology and KEGG pathway enrichment analyses showed that the DEGs were enriched in response to stress, secondary metabolism and metabolic pathways. Furthermore, 30 DEGs were assigned to stress response and possessed 2-15 E-box elements in their promoters, which could be potentially recognized and bound by ZmBES1/BZR1-5. Taken together, our results reveal that the ZmBES1/BZR1-5 transcription factor positively regulates salt and drought tolerance by binding to E-box to induce the expression of downstream stress-related genes. Therefore, our study contributes to the better understanding of BES1/BZR1 function in the stress response of plants.

Expression, Subcellular Localization, and Interactions of CPK Family Genes in Maize.

Calcium-dependent protein kinase (CPKs) is a key player in the calcium signaling pathway to decode calcium signals into various physiological responses. cDNA sequences of 9 ZmCPK genes were successfully cloned from all four phylogenetic groups in maize. qRT-PCR analysis showed the expression variation of these selected genes under abscisic acid (ABA) and calcium chloride (CaCl2) treatment. Due to the presence of N-myristoylation/palmitoylation sites, the selected ZmCPK members were localized in a plasma membrane. To clarify whether ZmCPK, a key player in calcium signaling, interacts with key players of ABA, protein phosphatase 2Cs (PP2Cs) and the SNF1-related protein kinase 2s (SnRK2s) and mitogen-activated protein kinase (MAPK) signaling pathways in maize, we examined the interaction between 9 CPKs, 8 PP2Cs, 5 SnRKs, and 20 members of the MPK family in maize by using yeast two-hybrid assay. Our results showed that three ZmCPKs interact with three different members of ZmSnRKs while four ZmCPK members had a positive interaction with 13 members of ZmMPKs in different combinations. These four ZmCPK proteins are from three different groups in maize. These findings of physical interactions between ZmCPKs, ZmSnRKs, and ZmMPKs suggested that these signaling pathways do not only have indirect influence but also have direct crosstalk that may involve the defense mechanism in maize. The present study may improve the understanding of signal transduction in plants.

Maize leaf-removal: A new agronomic approach to increase dry matter, flower number and seed-yield of soybean in maize soybean relay intercropping system.

Shading conditions adversely affect flower-number and pod-number of soybeans under maize-soybean relay-intercropping (MSR). Here we reveal that leaf-removal from maize-canopy improves the photosynthetically active radiation (PAR) transmittance and dry-matter production (DMP) of soybean (especially during the co-growth phase), and compensates the maize seed-yield loss by considerably increasing soybean seed-yield. In a two-year experiment with MSR, maize-plants were subjected to different leaf-removal treatments to increase the PAR-transmittance of soybean; removal of the topmost two-leaves (R2), four-leaves (R4), six-leaves (R6), with no-removal of leaves (R0). Leaf-removal treatments improved the PAR-transmittance, photosynthetic-rate, and morphological-characteristics of soybean under MSR. At 90 days after sowing, the dry-matter of pods, and seeds was increased by 25%, and 32%, respectively under R6 than R0. Importantly, enhanced PAR-transmittance and DMP under R6 enabled soybean to initiate a greater number of flowers 182.2 plant-1 compared to 142.7 plant-1 under R0, and it also decreased the flower-abscission (by 13%, from 54.9% under R0 to 47.6% under R6). These positive responses increased the pod-number by 49% and seed-number by 28% under R6 than R0. Overall, under R6, relay-intercropped soybean produced 78% of sole-soybean seed-yield, and relay-intercropped maize produced 81% of sole-maize seed-yield and achieved the land equivalent ratio of 1.59.

Growth and development of soybean under changing light environments in relay intercropping system.

BACKGROUND: Maize-soybean relay-intercropping (MSR) is a famous system of crop production in developing countries. However, maize shading under this system directly affects the light quality and intensity of soybean canopy. This is a challenging scenario in which to implement the MSR system, in terms of varieties selection, planting pattern, and crop management since the duration of crop resource utilization clearly differs. METHODS: Therefore, this experiment aimed to elucidate the effect of leaf excising treatments from maize top to fully clarify the needs and balance of light quality and intensity of intercrop-soybean under MSR in field conditions. The effects of different leaf excising treatments (T0, no removal of leaves; T2, removal of two topmost leaves; T4, removal of four topmost leaves; T6, removal of six topmost leaves from maize plants were applied at first-trifoliate stage (V1) of soybean) on photosynthetically active radiation transmittance (PART), red to far-red ratio (R:FR), morphological and photosynthetic characteristics and total biomass production at second-trifoliate stage (V2), fifth-trifoliate stage (V5), and flowering-stage (R1) of soybean were investigated through field experiments for 2-years under MSR. RESULTS: As compared to T0, treatment T6 increased the PART and R:FR ratio at soybean canopy by 77% and 37% (V2), 70% and 34% (V5), and 41% and 36% (R1), respectively. This improved light environment in T6 considerably enhanced the leaf area index, SPAD values and photosynthetic rate of soybean plants by 66%, 25% and 49% at R1, respectively than T0. Similarly, relative to control, T6 also increased the stem diameter (by 29%) but decreased the plant height (by 23%) which in turn significantly increased stem breaking strength (by 87%) by reducing the lodging rate (by 59%) of soybean plants. Overall, under T6, relay-cropped soybean produced 78% of sole soybean seed-yield, and relay-cropped maize produced 81% of sole maize seed-yield. Our findings implied that by maintaining the optimum level of PART (from 60% to 80%) and R:FR ratio (0.9 to 1.1), we can improve morphological and photosynthetic characteristics of soybean plants in MSR. Therefore, more attention should be paid to the light environment when considering the sustainability of MSR via appropriate planting pattern selection.

Physiological and Biochemical Responses of Pearl Millet (Pennisetum glaucum L.) Seedlings Exposed to Silver Nitrate (AgNO3) and Silver Nanoparticles (AgNPs).

: A rapid and continuous growth of silver nanoparticles (AgNPs) via their precursor "silver nitrate" (AgNO3) has increased their environmental risk because of their unsafe discharge into the surrounding environment. Both have damaging effects on plants and induce oxidative stress. In the present study, differential responses in the morpho-physiological and biochemical profiles of P. glaucum (L.) seedlings exposed to various doses of AgNPs and AgNO3 were studied. Both have forms of Ag accelerated the reactive oxygen species (ROS) production, which adversely affected the membrane stability as a result of their enhanced accumulation, and resulted in a significant reduction in growth, that is, root length, shoot length, fresh and dry biomass, and relative water content. AgNO3 possessed a higher degree of toxicity owing to its higher accumulation than AgNPs, and induced changes in the antioxidants' enzyme activity: superoxide dismutase (SOD), peroxidase (POD), catalases (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), and glutathione reductase (GR) activity, as well as proline content, total phenolic, and total flavonoids contents (TFCs) under all tested treatments (mM). A decline in photosynthetic pigments such as total chlorophyll content and carotenoid content and alterations in quantum yield (Fv/Fm), photochemical (qP), and non-photochemical quenching (NPQ) indicated the blockage of the electron transport chain (ETC), which led to a significant inhibition of photosynthesis. Interestingly, seedlings exposed to AgNPs showed less damaging effects on P. glaucum (L.) seedlings, resulting in relatively lower oxidative stress in contrast to AgNO3. Our results revealed that AgNO3 and AgNPs possessed differential phytotoxic effects on P. glaucum (L.) seedlings, including their mechanism of uptake, translocation, and action. The present findings may be useful in phytotoxic research to design strategies that minimize the adverse effects of AgNPs and AgNO3 on crops, especially in the agriculture sector.

Isolation and identification of a vegetative organ-specific promoter from maize.

To avoid the unregulated overexpression of the exogenous genes, specific or inducible expression is necessary for some exogenous genes in transgenic plants. But little is known about organ- or tissue-specific promoters in maize. In the present study, the expression of a maize pentatricopeptide repeat (PPR) protein encoding gene, GRMZM2G129783, was analyzed by RNA-sequencing data and confirmed by quantitative real time PCR. The results showed that the PPR GRMZM2G129783 gene specifically expressed in vegetative organs. Consequently, a 1830 bp sequence upstream of the start codon of the promoter for GRMZM2G129783 gene was isolated from maize genome (P 1830 ). To validate whether the promoter possesses the vegetative organ-specificity, the full-length and three 5'-end deletion fragments of P 1830 of different length (1387, 437, and 146 bp) were fused with glucuronidase (GUS) gene to generate promoter::GUS constructs and transformed into tobacco. The transient expression and fluorometric GUS assay in transgenic tobacco showed that all promoter could drive the expression of the GUS gene, the - 437 to - 146 bp region possessed some crucial elements for root-specific expression, and the shortest and optimal sequence to maintain transcription activity was 146 and 437 bp in length, respectively. These results indicate that the promoter of the PPR GRMZM2G129783 gene is a vegetative organ-specific promoter and will be useful in transgenic modification of commercial crops for moderate specific expression after further evaluation in monocotyledons.

Narrow-wide row planting pattern improves the light environment and seed yields of intercrop species in relay intercropping system.

Different planting patterns affect the light interception of intercrops under intercropping conditions. Here we revealed that narrow-wide-row relay-intercropping improves the light interception across maize leaves in wide rows (60cm) and narrow rows (40cm), accelerated the biomass production of intercrop-species and compensated the slight maize yield loss by considerably increasing the soybean yield. In a two-year experiment, maize was planted with soybean in different planting patterns (1M1S, 50:50cm and 2M2S, 40:60cm) of relay-intercropping, both planting patterns were compared with sole cropping of maize (M) and soybean (S). As compared to M and 1M1S, 2M2S increased the total light interception of maize leaves in wide rows (WR) by 27% and 23%, 20% and 10%, 16% and 9% which in turn significantly enhanced the photosynthetic rate of WR maize leaves by 7% and 5%, 12% and 9%, and 19% and 4%, at tasseling, grain-filling and maturity stage of maize, respectively. Similarly, the light transmittance at soybean canopy increased by 218%, 160% and 172% at V2, V5 and R1 stage in 2M2S compared with 1M1S. The improved light environment at soybean canopy in 2M2S considerably enhanced the mean biomass accumulation, and allocation to stem and leaves of soybean by 168%, and 131% and 207%, respectively, while it decreased the mean biomass accumulation, and distribution to stem, leaves and seed of maize by 4%, and 4%, 6% and 5%, respectively than 1M1S. Compared to 1M1S, 2M2S also increased the CR values of soybean (by 157%) but decreased the CR values of maize (by 61%). Overall, under 2M2S, relay-cropped maize and soybean produced 94% and 69% of the sole cropping yield, and the 2M2S achieved LER of 1.7 with net income of 1387.7 US $ ha-1 in 2016 and 1434.4 US $ ha-1 in 2017. Our findings implied that selection of optimum planting pattern (2M2S) may increase the light interception and influence the light distribution between maize and soybean rows under relay-intercropping conditions which will significantly increase the intercrops productivity. Therefore, more attention should be paid to the light environment when considering the sustainability of maize-soybean relay-intercropping via appropriate planting pattern selection.

The Influence of Light Intensity and Leaf Movement on Photosynthesis Characteristics and Carbon Balance of Soybean.

In intercropping systems shading conditions significantly impair the seed yield and quality of soybean, and rarely someone investigated the minimum amount of light requirement for soybean growth and development. Therefore, it is an urgent need to determine the threshold light intensity to ensure sustainable soybean production under these systems. An integrated approach combining morphology, physiology, biochemistry and genetic analysis was undertaken to study the light intensity effects on soybean growth and development. A pot experiment was set up in a growth chamber under increasing light intensity treatments of 100 (L100), 200 (L200), 300 (L300), 400 (L400), and 500 (L500) µmol m-2 s-1. Compared with L100, plant height, hypocotyl length, and abaxial leaf petiole angle were decreased, biomass, root:shoot ratio, and stem diameter were increased, extremum was almost observed in L400 and L500. Leaf petiole movement and leaf hyponasty in each treatment has presented a tendency to decrease the leaf angle from L500 to L100. In addition, the cytochrome content (Chl a, Chl b, Car), net photosynthetic rate, chlorophyll fluorescence values of F v/F m, F v ' / F m ' , ETR, ΦPSII, and qP were increased as the light intensity increased, and higher values were noted under L400. Leaf microstructure and chloroplast ultrastructure positively improved with increasing light intensity, and leaf-thickness, palisade, and spongy tissues-thickness were increased by 105, 90, and 370%, under L500 than L100. Moreover, the cross-sectional area of chloroplast (C) outer membrane and starch grains (S), and sectional area ratio (S:C) was highest under L400 and L500, respectively. Compared to L100, the content of starch granules increased by 35.5, 122.0, 157.6, and 145.5%, respectively in L400. The same trends were observed in the enzyme activity of sucrose-synthase, sucrose phosphate synthase, starch synthase, rubisco, phosphoenol pyruvate carboxykinase, and phosphoenol pyruvate phosphatase. Furthermore, sucrose synthesis-related genes were also up-regulated by increasing light intensity, and the highest seed yield and yield related parameters were recorded in the L400. Overall, these results suggested that 400 and 500 µmol m-2 s-1 is the optimum light intensity which positively changed the leaf orientation and adjusts leaf angle to perpendicular to coming light, consequently, soybean plants grow well under prevailing conditions.